| ObjectiveThe Ang2-siRNA plasmid chitosan magnetic nanoparticles prepared in the previous study group were injected into mice and rats through tail vein administration to observe the safety and toxicology of the composite microparticles.To establish the nude mice model of malignant melanoma and inject the composite microparticles into the mice through tail vein,then observe the targeting of the composite microparticles and inhibition of transplanted malignant melanoma angiogenesis and tumor growth,which provide new ways and research basis of targeted inhibition in angiogenesis and tumor growth of malignant melanoma.Methods1?The acute toxicology test of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesThe KM mice were used as experimental subjects for acute toxicology test,each group of mice was singly injected with 400ul five concentrations by tail vein(including 91.6 mg/kg,152.8 mg/kg,254.6 mg/kg,424.2 mg/kg,707.0 mg/kg)of Ang2-siRNA plasmid chitosan magnetic nanoparticles and physiological saline solution.The general condition of the mice was observed continuously for 2 weeks.The mice were killed 2 weeks later and histopathological examination,HE staining and Prussian staining of important organs were used to observe whether the organs were damaged.2?The experimental study on repeat-dose of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesThe SD rats were used as subjects for chronic toxicology experiment,which were divided into low-,medium-,and high-dose group(35.35 mg/kg,70.70 mg/kg,353.50 mg/kg)and control group.After 2 weeks of continuous injection of 1 ml of composite microparticles and physiological saline into the experimental and control groups of rats,the drug was discontinued on the 15th day,and observation was continued for 7 days.Observe and record the general performance and weigh changes of rats.Rats were executed on the 21st day and their eyeball blood were quickly taken for blood routine examination and biochemical examination.Histopathological examinations of important organs,HE staining and Prussian staining were used to observe whether the organs were damaged.3?The targeted study in vivo of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesAnimal models of transplantation tumor malignant melanoma in nude mice were constructed,and human malignant melanoma cells(A375 cells)in logarithmic growth phase were made into cell suspension,which were then inoculated into the right axillary subcutaneous tissue of nude mice.When the subcutaneous tumors grew to approximately 6×6 mm~2,the malignant melanoma nude mice were randomly divided into targeted group,non-targeted group and blank control group.The nude mice in the targeted group were injected with 400ul of Ang2-siRNA plasmid chitosan magnetic nanoparticles via tail vein injection,and the right temporal portion was placed in a stable magnetic field of 4000 GS for 60 minutes.The nude mice of the non-targeting group and control group were given 400ul of composite microparticles and physiological saline without magnetic field,60 minutes later each group of nude mice were killed.Then the tumor tissues of each group were taken for pathological examination.HE staining and Prussian blue staining were used to observe the distribution of microparticles in the nude mice.4?The study of inhibitory effect of Ang2-siRNA plasmid chitosan magnetic nanoparticles on growth of implanted malignant melanoma in nude miceThe transplanted tumor of malignant melanoma nude mice modles were randomly divided into control group and experimental group,10 in each group.The nude mice in control group were injected 400ul normal saline by tail vein without magnetic.The nude mice in experimental group were injected 400ul composite microparticles and under the 4000GS magnetic field cling right axillary subcutaneous tumor for 60 min,once every 3 days,total of 8 times.To observe and record the proliferation and volume changes of malignant melanoma transplanted tumors in each group,statistical analysis the volume difference,and draw the tumor growth curves of each group.Real-time fluorescence quantitative RT-PCR assay was used to detect the relative expression of Ang2 gene in the transplanted tumors of nude mice.The relationship between the expression of Ang2 gene and the degree of tumor cell proliferation was counted and analyzed.The microvessel density of transplanted tumors was detected by immunohistochemistry,statistical analysis the differences between the two groups and their relationship with tumor growth and proliferation.Tunel staining method was used to detect the apoptosis rate.Results1?The acute toxicology test of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesThere was no death in all mice after administration.There was no obvious general conditions changes of mice in each group after administration.Lung tissue pathology of nude mice in high-concentration group suggested signs of pulmonary congestion.No significant changes were observed in histopathological examinations of important organs in other groups.The mouse LD50 of composite microparticles was>707.0mg/kg,indicating that its acute toxicity was relatively low.2?The experimental study on repeat-dose of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesAfter the administration,there were no significant effects on the general activities,liver and kidney function in the rats of the groups.There was no obvious abnormality in the pathological examination of the main organs in the low dose group.The pulmonary pathological examination rats in the medium and high dose groups revealed chronic pulmonary congestion and fibrosis and there was no obvious abnormality in the other organs.The lowest dose in this experiment was 35.35mg/(kg.d)×14d,which was basically nontoxic to rats.Long-term application of the drug within the certain concentration range is safe.3?The targeted study in vivo of the Ang2-siRNA plasmid chitosan magnetic nanoparticlesThe animal model of human malignant melanoma transplanted tumor in nude mice was successfully constructed and no death occurred in each group of nude mice.Prussian blue staining in the control,non-targeted,and targeted groups in tumor tissue was negative,weakly positive,and strongly positive.The result indicates that after the Ang2-siRNA plasmid chitosan magnetic nanoparticles were injected into nude mice,they could be targeted and accumulated in the transplanted tumor tissue under the action of magnetic field.4?The study of inhibitory effect of Ang2-siRNA plasmid chitosan magnetic nanoparticles on growth of implanted malignant melanoma in nude miceNo death occurred after administration of tumor-bearing nude mice in each group.The average volume of transplanted tumors in the experimental group was1,200.81±267.32 mm~3,which was smaller than that of the black group(6312.01±914.62 mm~3),and the difference was statistically significant.Real-time fluorescence quantitative RT-PCR results showed that Ang2-siRNA plasmid chitosan magnetic nanoparticles inhibited Ang2 gene expression in the transplanted tumor by up to 37.33%.The MVD counts showed that the tumor microvessel density in the experimental group was 11.52±0.93/HP,which was lower than that in the black group(15.67±1.51/HP),and the difference was statistically significant.Tunel staining results showed that the apoptosis rate of the transplanted tumor cells in the experimental group was 5.35±1.81%,which was more than that of the black group(3.32±0.97%),and the difference was statistically significant.ConclusionThe Ang2-siRNA plasmid chitosan magnetic nanoparticles are safe to use within a certain dose range,and have the characteristics of targeted drug delivery system carrier.It can inhibit the occurrence and growth of transplanted malignant melanoma blood vessels in nude mice,and can inhibit the proliferation and development of malignant melanoma. |
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